Combination vaccine for intradermal administration

Abstract

To provide combination vaccines for swine.SOLUTION: Provided is a combination vaccine for protection against a pathogenic infection with porcine circo virus type 2 (PCV2) and Mycoplasma hyopneumoniae (Mhyo) comprising non-replicating immunogen of PCV2 and non-replicating immunogen of Mhyo. The vaccine is characterized in that it is an oil-in-water emulsion comprising squalane, vitamin E-acetate and silica. In another embodiment, the invention provides a combination vaccine for protection against a pathogenic infection with PCV2 and Mhyo by intradermal administration.SELECTED DRAWING: None

Description

Technical Field

[0001] General field of the invention The present invention relates to the field of veterinary vaccinology, namely, a combined vaccine for pigs . In particular, the present invention relates to a non-replicating immunogen of Porcine circovirus type 2 (PCV2) and a non-replicating immunogen of Mycoplasma · hyopneumoniae (Mhyo ) for protection against pathogenic infections caused by. The combined vaccine contains a non-replicating immunogen of PCV2 and a non-replicating immunogen of Mhyo. The vaccine is characterized by being an oil-in-water emulsion containing squalane, vitamin E- acetate and silica. In another embodiment, the present invention relates to a combined vaccine for protection against pathogenic infections caused by PCV2 and Mhyo by intradermal administration.

Background Art

[0002] Today's intensive pig farming heavily relies on veterinary pharmaceuticals to maintain animal health and enable operation. In addition to the optimization of feed and farm management systems, various treatments, namely, pharmaceuticals such as hormones or antibiotics, and vaccination against bacterial or viral pathogens are regularly used. Some of the most prominent diseases that affect pigs from an early age are caused by bacteria such as Mycoplasma hyopneumoniae and viruses such as Porcine circovirus type 2.

[0003] Mycoplasma hyopneumoniae (Mhyo) is a major factor causing enzootic pneumonia, a chronic respiratory disease in pigs that occurs worldwide. In particular, young piglets are It is susceptible to this highly contagious disease. The bacterium is relatively small, lacks a cell wall, and belongs to the genus Mycoplasma. These bacteria inhabit in a parasitic lifestyle on or inside the surface of host cells. The lung disease caused by Mhyo is mainly an immune-mediated pathological condition that leads to consolidated pneumonia. The bacterium colonizes the pulmonary ciliated epithelium, causes damage, and results in the loss of ciliary activity. Depending on the containment status and environmental stress, the most problematic effect of this disease is that it predisposes to various secondary infections of the porcine respiratory system by other bacterial and viral pathogens. This causes the so-called porcine respiratory complex infection (PRDC) that shows severe lung lesions. In addition to the discomfort of animals, enzootic pneumonia and PRDC result in significant economic losses to the pig farming industry due to the decline in performance in growth rate and feed conversion rate, as well as the costs for veterinary care and the use of antibiotics. Porcine circovirus type 2 (PCV2) is associated with postweaning multisystemic wasting syndrome (PMWS) observed in young pigs. Clinical signs and pathology were published in 1996 and

[0004] include progressive wasting, dyspnea, tachypnea, and sometimes jaundice and icterus. A new factor, different from the known PCV which was a natural contaminant of PK-15 cells, was named PCV2. PCV2 is a very small non-enveloped virus of the genus Circovirus. It contains a circular single-stranded DNA genome with two major genes. The ORF2 gene encodes a viral capsid protein of about 233 amino acids. The recombinantly expressed PCV2 ORF2 protein forms virus-like particles that are very effective as subunit vaccines.

[0005] There are various commercially available vaccines against Mhyo, and these are used routinely in most of the commercial pig farming industry. Generally, these vaccines contain non-replicating immunogens such as subunit proteins and / or bacterins (i.e., killed bacteria whether intact or not), and these are typically administered by parenteral injection. Some examples include RespiSure® (Zoetis), Ingelvac® M.hyo (Boehringer Ingelheim), and M+Pac® (Merck Animal Health). The normal vaccines for prophylactically treating animals, particularly pigs, against infection by PCV2 can be based on whole inactivated PCV2 virus as a (non-replicating) immunogen. Also, in the art, the capsid protein encoded by ORF2 (e.g., when recombinantly expressed) has been shown to be suitable as a subunit immunogen of PCV2 for use in appropriate vaccines. This can be understood in that this subunit appears in the body in the same way as the virus itself (it forms virus-like particles), and is essentially different only in that DNA and non-structural proteins are not present inside the capsid. In the art, several vaccines against PCV2 are commercially available. Porcilis® PCV (available from MSD Animal Health, Boxmeer, The Netherlands) is a vaccine for protecting pigs against porcine circovirus type 2 for use in pigs 3 weeks of age and older.

[0006] ​​​​​​​​​​​​​​​​Yes. When administered as a two-dose vaccine, the duration of immunity (DOI) is 2 weeks, which almost completely covers the fattening period of pigs. Ingelvac Cicro Flex® (available from Boehringer Ingelheim, Ingelhei m) is a vaccine used in pigs 2 weeks of age and older to protect pigs against porcine circovirus type 2. It is only registered as a single-dose (one-dose) vaccine. Circovac® (available from Merial, Lyon, Fra nce) is a vaccine used in pigs 3 weeks of age and older to protect pigs against porcine circovirus type 2. Suvaxyn® PCV( Zoeitis, Capelle a / d IJssel, The Netherlan ds) is a vaccine used in pigs 3 weeks of age and older to protect pigs against porcine circovirus type 2. Other PCV2 vaccines are described, for example, in WO 2007 / 028823, WO2007 / 094893 and WO2008 / 07691 5.

[0007] To reduce stress on animals and the labor and cost of breeders, several porcine vaccines are manufactured as combination vaccines. Specific examples include Ingelvac CircoFLEX and Ingelvac MycoFLE X (Boehringer), which can be mixed immediately before administration, Fostera® PCV MH (Zoetis), which combines the antigens of PCV2 and Mhyo, and Porcilis® PCV MHyo (MSD Animal Health).

[0008] An important component of vaccines containing non-replicating immunogens is the adjuvant. This provides immune stimulation to nonreplicating immunogens that are not immunogenic in the absence of a receptor. In veterinary medicine, the immune system triggers immune system pathways, but the underlying mechanisms are not fully understood. A wide variety of compounds are used as adjuvants in vaccines, including: Can also be used: mineral oils, such as Bayol® or Markol®, Mon tanide® or paraffin oil; non-mineral oils such as squalene, squalane or vegetable oils, e.g., ethyl oleate; aluminum salts, e.g., aluminum hydroxide or or aluminum phosphate; peptides, such as dimethylglycine or tuftsin; bacterial cells Cell wall components, such as lipid A and muramyl dipeptide; (synthetic) polymers, such as pleuropyridin; Nick, dextran, carbomer, pyran or saponin; cytokine; and thoronin. Stimulators of leukocyte-like receptors, such as immunostimulatory oligodeoxyribonuclease (ODR) receptors containing unmethylated CpG groups. Synucleotide; etc.

[0009] The main problem to be overcome when producing adjuvanted combination vaccines is the immune response or Interactions between various vaccine components that could adversely affect the safety or stability of the vaccine Such interactions can occur, for example, between the immunogens themselves. Some, such as Mhyo's bacterin, are very crude products. However, with regard to PCV2 and Mhyo, the art has not yet been able to identify the various types of these pathogens. It has already been shown that non-replicating immunogens can be combined in an effective vaccine. Moreover, adjuvants can inhibit or even damage vaccine immunogens. This is also recognized by the registration authorities that grant marketing authorizations. For example, the USDA enforces Rule 9 CFR 113.35 regarding the detection of virus-killing activity in inactivated vaccines containing live viruses. In addition, certain administration routes can have a significant impact on the safety of adjuvant compositions. An adjuvant may be safe when administered intramuscularly, but may cause unacceptable safety issues when administered subcutaneously. These potential problems in the development of complex combination vaccines are generally recognized. For example, refer to the publication "Note for guidance: requirements for combined veterinary products" (EMEA, 2000, CVMP / IWP / 52 / 97-FINAL) from the EMEA, and the publication "Guidance for Industry, for the evaluation of combination vaccines for preventable diseases: Production, Testing and Clinical Studies", Docket No. 97N-0029 from the Center for Biologies Evaluation and Research, U.S. Department of Health and Human Services, Food and Drug Administration since April 1997. These potential problems in the development of complex combination vaccines are generally recognized. For example, refer to the publication "Note for guidance: requirements for combined veterinary products" (EMEA, 2000, CVMP / IWP / 52 / 97-FINAL) from the EMEA, and the publication "Guidance for Industry, for the evaluation of combination vaccines for preventable diseases: Production, Testing and Clinical Studies", Docket No. 97N-0029 from the Center for Biologies Evaluation and Research, U.S. Department of Health and Human Services, Food and Drug Administration since April 1997. These potential problems in the development of complex combination vaccines are generally recognized. For example, refer to the publication "Note for guidance: requirements for combined veterinary products" (EMEA, 2000, CVMP / IWP / 52 / 97-FINAL) from the EMEA, and the publication "Guidance for Industry, for the evaluation of combination vaccines for preventable diseases: Production, Testing and Clinical Studies", Docket No. 97N-0029 from the Center for Biologies Evaluation and Research, U.S. Department of Health and Human Services, Food and Drug Administration since April 1997. These potential problems in the development of complex combination vaccines are generally recognized. For example, refer to the publication "Note for guidance: requirements for combined veterinary products" (EMEA, 2000, CVMP / IWP / 52 / 97-FINAL) from the EMEA, and the publication "Guidance for Industry, for the evaluation of combination vaccines for preventable diseases: Production, Testing and Clinical Studies", Docket No. 97N-0029 from the Center for Biologies Evaluation and Research, U.S. Department of Health and Human Services, Food and Drug Administration since April 1997. These potential problems in the development of complex combination vaccines are generally recognized. For example, refer to the publication "Note for guidance: requirements for combined veterinary products" (EMEA, 2000, CVMP / IWP / 52 / 97-FINAL) from the EMEA, and the publication "Guidance for Industry, for the evaluation of combination vaccines for preventable diseases: Production, Testing and Clinical Studies", Docket No. 97N-0029 from the Center for Biologies Evaluation and Research, U.S. Department of Health and Human Services, Food and Drug Administration since April 1997.

[0010] These potential problems in the development of complex combination vaccines are generally recognized. For example, refer to the publication "Note for guidance: requirements for combined veterinary products" (EMEA, 2000, CVMP / IWP / 52 / 97-FINAL) from the EMEA, and the publication "Guidance for Industry, for the evaluation of combination vaccines for preventable diseases: Production, Testing and Clinical Studies", Docket No. 97N-0029 from the Center for Biologies Evaluation and Research, U.S. Department of Health and Human Services, Food and Drug Administration since April 1997. These potential problems in the development of complex combination vaccines are generally recognized. For example, refer to the publication "Note for guidance: requirements for combined veterinary products" (EMEA, 2000, CVMP / IWP / 52 / 97-FINAL) from the EMEA, and the publication "Guidance for Industry, for the evaluation of combination vaccines for preventable diseases: Production, Testing and Clinical Studies", Docket No. 97N-0029 from the Center for Biologies Evaluation and Research, U.S. Department of Health and Human Services, Food and Drug Administration since April 1997. These potential problems in the development of complex combination vaccines are generally recognized. For example, refer to the publication "Note for guidance: requirements for combined veterinary products" (EMEA, 2000, CVMP / IWP / 52 / 97-FINAL) from the EMEA, and the publication "Guidance for Industry, for the evaluation of combination vaccines for preventable diseases: Production, Testing and Clinical Studies", Docket No. 97N-0029 from the Center for Biologies Evaluation and Research, U.S. Department of Health and Human Services, Food and Drug Administration since April 1997. These potential problems in the development of complex combination vaccines are generally recognized. For example, refer to the publication "Note for guidance: requirements for combined veterinary products" (EMEA, 2000, CVMP / IWP / 52 / 97-FINAL) from the EMEA, and the publication "Guidance for Industry, for the evaluation of combination vaccines for preventable diseases: Production, Testing and Clinical Studies", Docket No. 97N-0029 from the Center for Biologies Evaluation and Research, U.S. Department of Health and Human Services, Food and Drug Administration since April 1997. These potential problems in the development of complex combination vaccines are generally recognized. For example, refer to the publication "Note for guidance: requirements for combined veterinary products" (EMEA, 2000, CVMP / IWP / 52 / 97-FINAL) from the EMEA, and the publication "Guidance for Industry, for the evaluation of combination vaccines for preventable diseases: Production, Testing and Clinical Studies", Docket No. 97N-0029 from the Center for Biologies Evaluation and Research, U.S. Department of Health and Human Services, Food and Drug Administration since April 1997. These potential problems in the development of complex combination vaccines are generally recognized. For example, refer to the publication "Note for guidance: requirements for combined veterinary products" (EMEA, 2000, CVMP / IWP / 52 / 97-FINAL) from the EMEA, and the publication "Guidance for Industry, for the evaluation of combination vaccines for preventable diseases: Production, Testing and Clinical Studies", Docket No. 97N-0029 from the Center for Biologies Evaluation and Research, U.S. Department of Health and Human Services, Food and Drug Administration since April 1997. These potential problems in the development of complex combination vaccines are generally recognized. For example, refer to the publication "Note for guidance: requirements for combined veterinary products" (EMEA, 2000, CVMP / IWP / 52 / 97-FINAL) from the EMEA, and the publication "Guidance for Industry, for the evaluation of combination vaccines for preventable diseases: Production, Testing and Clinical Studies", Docket No. 97N-0029 from the Center for Biologies Evaluation and Research, U.S. Department of Health and Human Services, Food and Drug Administration since April 1997. These potential problems in the development of complex combination vaccines are generally recognized. For example, refer to the publication "Note for guidance: requirements for combined veterinary products" (EMEA, 2000, CVMP / IWP / 52 / 97-FINAL) from the EMEA, and the publication "Guidance for Industry, for the evaluation of combination vaccines for preventable diseases: Production, Testing and Clinical Studies", Docket No. 97N-0029 from the Center for Biologies Evaluation and Research, U.S. Department of Health and Human Services, Food and Drug Administration since April 1997. These potential problems in the development of complex combination vaccines are generally recognized. For example, refer to the publication "Note for guidance: requirements for combined veterinary products" (EMEA, 2000, CVMP / IWP / 52 / 97-FINAL) from the EMEA, and the publication "Guidance for Industry, for the evaluation of combination vaccines for preventable diseases: Production, Testing and Clinical Studies", Docket No. 97N-0029 from the Center for Biologies Evaluation and Research, U.S. Department of Health and Human Services, Food and Drug Administration since April 1997. These potential problems in the development of complex combination vaccines are generally recognized. For example, refer to the publication "Note for guidance: requirements for combined veterinary products" (EMEA, 2000, CVMP / IWP / 52 / 97-FINAL) from the EMEA, and the publication "Guidance for Industry, for the evaluation of combination vaccines for preventable diseases: Production, Testing and Clinical Studies", Docket No. 97N-0029 from the Center for Biologies Evaluation and Research, U.S. Department of Health and Human Services, Food and Drug Administration since April 1997. These potential problems in the development of complex combination vaccines are generally recognized. For example, refer to the publication "Note for guidance: requirements for combined veterinary products" (EMEA, 2000, CVMP / IWP / 52 / 97-FINAL) from the EMEA, and the publication "Guidance for Industry, for the evaluation of combination vaccines for preventable diseases: Production, Testing and Clinical Studies", Docket No. 97N-0029 from the Center for Biologies Evaluation and Research, U.S. Department of Health and Human Services, Food and Drug Administration since April 1997. These potential problems in the development of complex combination vaccines are generally recognized. For example, refer to the publication "Note for guidance: requirements for combined veterinary products" (EMEA, 2000, CVMP / IWP / 52 / 97-FINAL) from the EMEA, and the publication "Guidance for Industry, for the evaluation of combination vaccines for preventable diseases: Production, Testing and Clinical Studies", Docket No. 97N-0029 from the Center for Biologies Evaluation and Research, U.S. Department of Health and Human Services, Food and Drug Administration since April 1997. Both substances are warning about the influence of interference on the effectiveness and safety of vaccines when combining an immunogen and an adjuvant.

[0011] Therefore, it is difficult to develop a combined vaccine that induces an effective immune response against complex combinations of immunogens related to multiple species of pathogens. Furthermore, the combined vaccine must be safe for use in animals. That is, it must not cause any serious side effects such as fever, local swelling, and loss of appetite. Also, more practical characteristics are important, and the combined vaccine should ideally be capable of economical production, be sufficiently stable during formulation (prescription) and storage, and be able to perform potency testing methods for each immunogen in the presence of other immunogens. In general, it is known that vaccinating animals is not easy, especially when the vaccination is with an adjuvant-containing combined vaccine, and experiments are required to determine safety and effectiveness.

[0012] Overall, vaccinating against multiple pathogens is not simple, especially when the vaccination is with an adjuvant-containing combined vaccine, and experiments are required to determine safety and effectiveness.

[0013] Therefore, it is necessary to provide an effective and safe combined vaccine against diseases related to infections caused by PCV2 and Mhyo, overcoming one or more drawbacks in the prior art.

[0014] In vaccinating a large number of animals, it is important to reduce stress for both the animals and the people vaccinating them. Furthermore, vaccination by the normal intramuscular route often involves pain and stress in animals, increasing the risk of side effects and infections. ​​​​​​​​​​​​is involved. One possibility to overcome the problems associated with intramuscular administration is intradermal vaccination (also referred to as intradermal inoculation). Intradermal vaccination allows for administration over a wider range of sites in animals, increasing the freedom of the user. This is particularly useful when vaccinating large populations of pigs. This is because it allows for rapid and non-invasive application, reducing stress for both the pigs and the handlers.

[0015] The systemic and respiratory local immune responses induced by subcutaneous administration of a commercially available inactivated Mycoplasma hyopneumoniae whole cell vaccine (Porcilis( registered trademark) MHYO ID ONCE - MSD Animal Health) have been compared with those of two commercially available vaccines administered by the intramuscular route and a negative control, as described in P. Martelli et al., Vet Microbiol. 2014;168(2 - 4):357 - 64, and it has been shown that intradermal administration of an adjuvant - containing Mhyo bacterin induces both systemic and mucosal immune responses.

[0016] In particular, intradermal administration has the advantage that it can be carried out by a needle - free vaccination device such as the IDAL (registered trademark) vaccination device (available from MSD Animal Health, Boxmeer, The Netherlands). The "intradermal" administration itself should not be equated with "needle - free" administration. The vaccine can only be delivered (at least partially) intradermally if the needle - free device is "designed for intradermal vaccination". Needle - free intradermal administration is less invasive than needle injection, has fewer systemic side effects in It induces an immune response. It also reduces the risk of needles transmitting disease among pigs during vaccination.

[0017] However, it is difficult to provide a safe and effective vaccine suitable for intradermal administration. This is because the volume of the vaccine needs to be very small, typically in the range of about 0.1 - 0.5 ml. Therefore, the immunogen and other components of the vaccine (e.g., possible adjuvants) need to be highly concentrated, which increases the risk of interactions between the various vaccine components. As mentioned above, vaccines combining immunogens from PCV2 and Mhyo are commercially available. However, these mixed vaccines are for intramuscular administration and are not suitable for intradermal administration. SUMMARY OF THE INVENTION

[0018] OBJECT OF THE INVENTION It was an object to provide a mixed vaccine against diseases associated with infections by PCV2 and Mhyo, suitable for intradermal administration. In particular, it was an object to provide a safe and effective vaccine that can be used for the prophylactic treatment of animals against infections by PCV2 and Mhyo.

[0019] SUMMARY OF THE INVENTION Surprisingly, by devising a specific adjuvant that enables a mixed vaccine containing a non - replicating immunogen of porcine circovirus type 2 (PCV2) and a non - replicating immunogen of Mycoplasma hyopneumoniae (Mhyo) (where the mixed vaccine is an oil - in - water emulsion containing squalene, vitamin E - acetate, and silica), these objects can be achieved, and as a result, it has been found that one or more drawbacks of the prior art can be overcome. 。

[0020] In particular, surprisingly, a mixed vaccine comprising a non-replicating immunogen of PCV2 and a non-replicating immunogen of Mhyo and comprising squalane, vitamin E-acetate and silica in an oil-in-water emulsion (i.e., the continuous phase of the emulsion is aqueous and within it, a hydrophobic liquid which is the discontinuous phase is dispersed, and within the latter phase itself a second or further phase may be dispersed) has been found to be usable by intradermal administration in the safe prophylactic treatment of animals against infection by PCV2 and infection by Myho.

[0021] The present invention also resides in the adjuvant composition itself, in particular in an adjuvant composition for formulating a non-live vaccine, where the composition is an oil-in-water emulsion comprising squalane, vitamin E-acetate and silica.

[0022] Definitions A "mixed vaccine" is a vaccine comprising immunogens from multiple microbial species. The mixed vaccine according to the present invention comprises at least immunogens from porcine circovirus type 2 and Mycoplasma hyopneumoniae. Thus, the mixed vaccine according to the present invention may also be colloquially referred to as a vaccine "against" PCV2 and Mhyo.

[0023] A "vaccine" generally means a pharmaceutical composition which can be safely administered to a target animal such as a pig and which can induce a protective immunity in that animal against pathogenic microorganisms. A vaccine typically comprises an immunologically active ingredient and a pharmaceutically acceptable carrier. An "immunologically active" The "active ingredient" is one or more immunogenic molecules, for example, non-replicating immunogens from PCV2 and Mhyo. These are recognized by the immune system of the target animal and induce a protective immune response.

[0024] Vaccines are generally effective in reducing the severity of infection, for example, by reducing the number of pathogens or by shortening the period of replication of the pathogens in the host animal. In addition, or perhaps as a result, vaccines are generally effective in reducing or ameliorating the (clinical) symptoms of diseases that may be caused by such infection or replication, or by the animal's response to

[0025] such infection or replication. A "non-replicating immunogen" of a pathogen is any substance or compound corresponding to the pathogen, excluding the whole live replicating pathogen (either in its attenuated wild-type form or any other), against which an immune response is induced, and as a result of this immune response, one or more of the corresponding virulent pathogens or their pathogenic factors are recognized by the host's immune system and ultimately at least partially neutralized. Typical examples of non-replicating immunogens are killed whole pathogens (this term includes these

[0026] pathogens in their lysed form), as well as subunits of these pathogens, such as capsid proteins, surface-expressed molecules (e.g., recombinant expressed proteins or lipopolysaccharides) and secreted molecules, such as toxins. Reduce the burden on the host after challenge and, optionally, treatment by the pathogen Help prevent or ameliorate one or more clinical symptoms resulting from post-infection.

[0027] Embodiments of the invention In a first embodiment, the invention is a mixed vaccine comprising a non-replicating immunogen of PCV2 and a non-replicating immunogen of Mhyo, characterized in that it is an oil-in-water emulsion comprising squalane, vitamin E-acetate and saponin. The mixed vaccine according to the invention relates to a mixed vaccine which is an oil-in-water emulsion comprising squalane, vitamin E-acetate and saponin.

[0028] Each of the "non-replicating immunogens" in the mixed vaccine according to the invention can be of a single type, or can be of a plurality of types derived from, for example, one or more strains of each pathogen. In the case of the present invention, the non-replicating immunogen of PCV2 is preferably inactivated whole PCV2 virus. Even more preferred is the use of the ORF2 protein as a subunit, which is typically obtained from a recombinant expression system and is transported and expressed by replicon particles. PCV2 ORF2 is expressed and recovered by recombinant baculovirus in insect cell culture. The replicon particles are defective virus particles such as, for example, alphavirus particles developed by Alpha Vax. The parental PCV2 of the expressed ORF2 sequence can be any of PCV2 serotypes a, b, c or d, or can be derived from one or more chimeras of these serotypes. The non-replicating immunogen of Mycoplasma hyopneumoniae typically comprises killed whole Mycoplasma hyopneumoniae, i.e., killed Mhyo bacterin. Mhyo bacterin is, for example, a bacterin obtained by inactivating Mhyo with a chemical agent or heat treatment. The bacterin can be a monovalent bacterin comprising only one strain of Mhyo, or a multivalent bacterin comprising two or more strains of Mhyo. The strain of Mhyo in the bacterin can be any of the known Mhyo strains, such as, for example, the J strain, the 7448 strain or the 232 strain. The bacterin can be prepared by methods known in the art,

[0029] The non-replicating immunogen of Mycoplasma hyopneumoniae typically comprises killed whole Mycoplasma hyopneumoniae, i.e., killed Mhyo bacterin. Mhyo bacterin is, for example, a bacterin obtained by inactivating Mhyo with a chemical agent or heat treatment. The bacterin can be a monovalent bacterin Phosphorus is preferably derived from strain 11 or strain J.

[0030] An "oil-in-water emulsion" is an emulsion having an aqueous continuous phase, in which a hydrophobic liquid, which is a discontinuous phase, is dispersed inside the aqueous continuous phase, and a second or further phase may be dispersed inside the latter phase itself. Such an emulsion can be formed by selecting an appropriate type and concentration of emulsifier. Methods and apparatuses for the production of oil-in-water emulsions used as vaccines are well known in the art and are described, for example, in handbooks such as "Remington: the science and practice of pharmacy" (2000, Lippincot, USA, ISBN: 683306472) and "Veterinary vaccinology" (edited by P. Pastoret et al., 1997, Elsevier, Amsterdam, ISBN 0444819681). In the present invention, the outer aqueous phase can contain a non-replicating immunogen from PCV2 and Myho as well as silica, and the oil phase can contain squalane and vitamin E acetate. The mixed vaccine according to the present invention has been found to be very effective, safe and stable when produced as an oil-in-water emulsion. Embodiments and preferred forms for the production of the oil-in-water emulsion for the mixed vaccine according to the present invention are described hereinafter in this specification.

[0031]

[0032] The mixed vaccine according to the present invention has been found to be very effective, safe and stable when produced as an oil-in-water emulsion. Embodiments and preferred forms for the production of the oil-in-water emulsion for the mixed vaccine according to the present invention are described hereinafter in this specification.

[0033] "Squalane" is a non-mineral oil, shark liver oil, hexamethyltetracosane or per It is also called hydro squalene. This should not be confused with squalene (CAS number 111 - 02 - 4), which is a polyunsaturated C30 oil and can be metabolized as a compound in the cholesterol pathway. However, squalane is the fully hydrogenated form of squalene and is thus less prone to oxidation. Therefore, squalane can move from the injection site (and thus it "disappears" from the injection site), and thus it has sometimes been shown to be "metabolizable", but in fact it is an inert and non - metabolizable oil (it only physically moves from the injection site and is not metabolized). Initially, the precursor of squalane was obtained from shark liver, but due to environmental concerns, this has been replaced by other natural resources such as olive oil and chemical synthesis. Therefore, the definition of squalane includes natural, synthetic or semi - synthetic forms or mixtures thereof. Squalane is commercially available in various purities. For example, it can be obtained from vegetables, Worlee (squalane, vegetable) or Croda (Pripure squalane), or as a synthetic product from, for example, Kuraray (squalane - PE). In the case of the present invention, high - purity squalane is preferred, preferably with a purity exceeding 75%, more preferably with a purity exceeding 80, 90 or even 95% (the latter being more preferred). Squalane in the mixed vaccine according to the present invention is typically present in an amount of 1 - 15% w / v of the vaccine. More preferably, squalane is 3 - 12% w / v of the vaccine.

[0034] Originally, the precursor of squalane was obtained from shark liver. However, due to environmental concerns, this has been replaced by other natural resources such as olive oil and chemical synthesis. Therefore, the definition of squalane includes natural, synthetic or semi - synthetic forms or mixtures thereof. Squalane is commercially available in various purities. For example, it can be obtained from vegetables, Worlee (squalane, vegetable) or Croda (Pripure squalane), or as a synthetic product from, for example, Kuraray (squalane - PE). In the case of the present invention, high - purity squalane is preferred, preferably with a purity exceeding 75%, more preferably with a purity exceeding 80, 90 or even 95% (the latter being more preferred).

[0035] Squalane in the mixed vaccine according to the present invention is typically present in an amount of 1 - 15% w / v of the vaccine. More preferably, squalane is 3 - 12% w / v of the vaccine. ​​​​​​​​​​Or even 5 to 9% w / v (the latter being more preferred), for example, 5%, 6%, 7%, 8% or 9% w / v. Most preferably, squalane is present in an amount of about 6 .8% w / v.

[0036] Thus, in one embodiment of the mixed vaccine according to the present invention, the vaccine contains squalane in an amount of 1 to 15% w / v.

[0037] "Vitamin E-acetate" is the acetate ester of vitamin E (tocopherol), which may be derived from plant materials such as seeds, nuts, fruits or leaves or fats, but can also be produced synthetically. Some alternative names include tocopheryl acetate (tocopherol acetate ester) or alpha-tocopherol-acetate (alpha tocopherol acetate ester). The definition of vitamin E-acetate includes natural, synthetic or semi-synthetic forms or mixtures thereof. Vitamin E-acetate is commercially available in various purities. The vitamin E-acetate used in the mixed vaccine according to the present invention is the racemate of the chemical substance having the CAS number 7695-91-2, DL-al pha-tocopherol-acetate.

[0038] The vitamin E-acetate in the mixed vaccine according to the present invention is typically present in an amount of 2 to 20% w / v of the vaccine. More preferably, vitamin E-acetate is present in an amount of 4 to 16% w / v or 6 to 10% w / v (the latter being more preferred) of the vaccine, for example 6%, 7%, 8%, 9% or 10% w / v. Most preferably, vitamin E-acetate is present in an amount of about 8% w / v of the vaccine. ​​​

[0039] Thus, in one embodiment of the combination vaccine according to the invention, the vaccine comprises 2 to 5 Contains Vitamin E-acetate in an amount of 20% w / v.

[0040] "Silica" is silicon dioxide. Silica is used in adjuvant compositions It is widely described and generally referred to as pharmaceutical grade silica. All pharmaceutical Grades of silica have in common that they are colloidal silicon dioxide, and are used in the pharmaceutical industry This type of silica has been used for almost 50 years. and hydrophobic (e.g., methylated), crystalline or amorphous (e.g., fumed silica F) and various granulation ratios are available, all of which have the potential to be beneficial in adjuvant compositions. Commonly used. Examples of silica types preferably used in the adjuvant composition Examples of the silica include amorphous silica (which can be hydrophilic or hydrophobic, but is not limited to the silica used in the present invention). In the case where the amorphous silica is used, it is preferably hydrophilic. Silica is a fumed silica, which is produced in a flame and is also known as pyrogenic silica. It is known that the silica nanoparticles consist of minute droplets of amorphous silica, which are divided into branched three-dimensional secondary particles. They fuse together and then agglomerate into tertiary particles.

[0041] The silica used in the mixed vaccine of the present invention is 100 to 700 μm 2 / gram, more Preferably 300-500m 2 / g, and even more preferably 350-410m 2 / Gra m, most preferably about 395±25 m 2 The particle size may be 100 / g. The product is determined by methods known in the art, such as calculations using the Brunauer nitrogen adsorption method (Brunauer, S. et al., J .Am.Chem.Soc., 60, 309 (1938)). It can be determined by methods known in the art, such as calculations using the Brunauer nitrogen adsorption method (Brunauer, S. et al., J

[0042] Such products are commercially available, for example, under the trade names Aerosil® or Aeroperl® (in these trade names, a large number of variants such as different surface areas, hydrophobicity and hydrophilicity, crystallinity or amorphousness are available ). As an example, Aerosil® 380 by Evonik Resource Efficiency GmbH, Germany or S5130 by Sigma-A ldrich (having a particle size of about 0.007 μm, a pH of 3.7 - 4.5 (4% dispersion) and a tap density of about 50 g / l) can be mentioned. ldrich (having a particle size of about 0.007 μm, a pH of 3.7 - 4.5 (4% dispersion) and a tap density of about 50 g / l) can be mentioned. ). As an example, Aerosil® 380 by Evonik Resource Efficiency GmbH, Germany or S5130 by Sigma-A

[0043] The silica in the mixed vaccine according to the invention is typically present in an amount of 0.02 - 2% w / v of the vaccine. More preferably, the silica is present in an amount of 0.05 - 1.0% w / v or even 0.1 - 0.4% w / v (the latter being more preferred), for example, 0.1 %, 0.2%, 0.3% or 0.4% w / v. Most preferably, the silica is present in an amount of about 0.2% w / v of the vaccine.

[0044] Thus, in embodiments of the mixed vaccine according to the invention, the vaccine contains silica in an amount of 0.02 - 2% w / v.

[0045] The mixed vaccine of the invention typically contains a pharmaceutically acceptable carrier, preferably water . Preferably, the water is high-purity water, such as redistilled water, precision-filtered water or reverse osmosis water. More preferably, the water is water for injection, which is sterile and substantially free of pyrogens.

[0046] An advantageous feature of vaccines based on oil-in-water emulsions is that the immunogen is usually present in the aqueous phase . This means that methods and techniques that are not in themselves suitable for maintaining the quality or viability of vaccine immunogens are used, for example, high-energy emulsification is used at high temperatures, and the oil phase is prepared separately and can be emulsified in water. This produces an oil emulsion for the present invention, which is an oil-in-water emulsion of squalane, vitamin E-acetate and silica in water. To produce the combined vaccine according to the present invention, an aqueous phase containing an immunogen and silica is gently mixed with an oil emulsion containing other adjuvants at room temperature. The mixing of these two compositions causes dilution of each of them. Therefore, each of them needs to be prepared as an intermediate composition in which the concentrations of the various components are higher than those in the final vaccine by a factor equal to the dilution factor applied. Typically, the aqueous phase and the oil emulsion can be mixed in any volume ratio between 10:90 and 90:10. The combined vaccine according to the present invention preferably comprises the aqueous phase and the oil emulsion described together in a volume ratio of 20:80 to 80:20. Thus, in one embodiment the combined vaccine according to the present invention is produced from a mixture of an aqueous phase and an oil emulsion in a volume ratio of 20:80 to 80:20. Preferably, the volume ratio is 30:70 to 70:30, 40:

[0047] Mixing of these two compositions causes dilution of each of them. Thus, each of them needs to be prepared as an intermediate composition in which the concentrations of the various components are higher than those in the final vaccine by a factor equal to the dilution factor applied. Typically, the aqueous phase and the oil emulsion can be mixed in any volume ratio between 10:90 and 90:10. The combined vaccine according to the present invention preferably comprises the aqueous phase and the oil emulsion described together in a volume ratio of 20:80 to 80:20. Thus, in one embodiment the combined vaccine according to the present invention is produced from a mixture of an aqueous phase and an oil emulsion in a volume ratio of 20:80 to 80:20. Preferably, the volume ratio is 30:70 to 70:30, 40:

[0048] The combined vaccine according to the present invention preferably comprises the aqueous phase and the oil emulsion described together in a volume ratio of 20:80 to 80:20. Thus, in one embodiment the combined vaccine according to the present invention is produced from a mixture of an aqueous phase and an oil emulsion in a volume ratio of 20:80 to 80:20. Preferably, the volume ratio is 30:70 to 70:30, 40: the combined vaccine according to the present invention is produced from a mixture of an aqueous phase and an oil emulsion in a volume ratio of 20:80 to 80:20. Preferably, the volume ratio is 30:70 to 70:30, 40: 60 or 50:50. A volume ratio of 60 to 60:40; or even about 50:50 (the latter being more preferred). .

[0049] Obviously, when the mixing ratio of the aqueous phase and the oil-based emulsion is about 50:50, each of these two compositions should contain the various components in an amount or concentration that is twice as high as that desired in the final vaccine formulation produced by mixing the two intermediate compositions. .

[0050] In a preferred embodiment, the oil-based emulsion for the present invention is produced using an emulsifier having an HLB value (hydrophilic-lipophilic balance) of 8 to 20. A preferred emulsifier is polysorbate 80.

[0051] Polysorbate 80 refers to a chemical substance having the CAS number 9005-65-6 (also referred to as polyoxy ethylene sorbitan monooleate). It has an HLB value of about 15 and is widely commercially available, for example, as Tween 80.

[0052] Preferably, polysorbate 80 is present in the mixed vaccine according to the present invention in an amount of 0. 5 to 10% w / v of the vaccine. More preferably, polysorbate 80 is present in the vaccine in an amount of 0.7 to 7% w / v, 1.0 to 5% or even 2 to 4% w / v (the latter being more preferred). Most preferably, polysorbate 80 is present in the vaccine in an amount of about 3.2% w / v.

[0053] Thus, in one embodiment, the mixed vaccine according to the present invention contains polysorbate 80 in an amount of 0.5 to 10% w / v.

[0054] ​The oil-in-water emulsion for the present invention can be produced on any scale using any suitable homogenizing device such as a Microfluidizer™, Sil verson™, Ultra Turrax™ or a DispaX reactor - (IKA). Those skilled in the art can carry out and optimize such an emulsification process to control the size of the particles of the dispersed phase (in this case the oily adjuvant). Together with the choice of the type and concentration of the emulsifier, this controls the pharmaceutical properties of the emulsion and also its st ability. The main parameters of the emulsification process itself are the energy input (power and r pm), temperature, duration and number of repetition cycles. Details of the embodiments of the emulsification process are given below.

[0055]

[0056]

[0057] The size of the particles of the dispersed phase is preferably very small. When the diameter of the particles of the dispersed phase is less than about 1 micrometer, such an emulsion is generally referred to as a "submicron emulsion ". In one embodiment of the oil-in-water emulsion of the mixed vaccine according to the present invention, the emulsion is a submicron emulsion.

[0057] nstruments) and is expressed in nm of D50 as determined when using a Mastersizer® (Malvern I . Particle size measurements can be performed on (concentrated) oily emulsions or mixed vaccines. For the purposes of the present invention, the refractive index of the oil phase particles is 1.48. The size analysis report of Malvern Mastersizer represents D50 as D(0.50). Therefore, in one embodiment of the submicron water-in-oil emulsion of the mixed vaccine according to the present invention, the oil droplets have a D50 of 500 nm or less, preferably, the D50 is 250 nm or less. More preferably, the D50 is 150 nm or less.

[0058] There are a number of methods available for producing such submicron emulsions, typically high-energy emulsification processes are used, such as high-pressure homogenizers, rotor- stator (dynamic and static blade) devices, mixers (blenders), ultrasonic, microporous membranes or microchanneling devices are used.

[0059] A preferred process for high-energy emulsification for the purposes of the present invention is the use of a high-pressure homogenizer, preferably a Microfluidizer™ (Microfluidics). Typically, three passes at a pressure of 500 - 1500 bar (i.e., 7000 - 22000 psi) are sufficient. The emulsion thus produced typically has dispersed phase particles with a D50 of 500 nm or less and has a narrow size distribution. In the case of the present invention, the dispersed phase is droplets of an oily adjuvant.

[0060] Typically, emulsions having such very fine-sized particles of the dispersed phase are produced in several steps. In this way, an initial relatively coarse oily emulsion is Manufactured by ruggy mixing and then subjected to one or more subsequent high energy treatments. Next, a "microfluidised" oil-in-water emulsion containing an adjuvant and optionally an emulsifier in water is combined with an aqueous phase containing an immunogen to obtain the mixed vaccine according to the invention. For reasons of product consistency and quality, not only the median particle size but also the spread of the particle size (also known as the size distribution) can be advantageously monitored and controlled. The size distribution of the oil droplets in the submicron oil-in-water emulsion of the mixed vaccine according to the invention is preferably relatively

[0061] narrow. An indicator of the particle size distribution is D90 of the cumulative particle size distribution. Thus, in one embodiment of the submicron oil-in-water emulsion of the mixed vaccine according to the invention, the oil droplets have a D90 of less than 900 nm, and a more preferred D90 is less than 500 nm, less than 400 nm or even less than 300 nm (the latter being more preferred).

[0062] Most preferably, D90 is about 150 - 250 nm. One advantage of an emulsion having such a small particle size and distribution is that it can then be sterilized by filtration without significant loss of material. This is because typical sterilizing filters have a pore size of about 0.2 micrometers. Such

[0063] filtration sterilization avoids the need for other sterilization methods such as heating, chemicals or irradiation that can damage the quality of the components of the oil-in-water emulsion. Accordingly, the mixed vaccine according to the invention typically consists of PCV2 and Mhyo from and avoids

[0064] and A non-replicating immunogen is included in an amount capable of inducing a protective immune response against those related diseases in animal targets as described above. in an amount capable of inducing a protective immune response against those related diseases in animal targets as described above.

[0065] Those skilled in the art of the present invention can, for example, monitor the immunological response after vaccination or after challenge infection, for example, by monitoring target disease symptoms, clinical scores or by re-isolating the pathogen and comparing these results with the vaccination-challenge responses seen in mock-vaccinated animals to determine the effectiveness of the combination vaccines according to the present invention. It is fully possible to determine the effectiveness of the combination vaccines according to the present invention.

[0066] As one indicator, the amount of immunogen used in the combination vaccines according to the present invention can be based on that used in each monovalent or combination vaccine containing these immunogens. For example, the combination vaccines according to the present invention can contain 1-150 μg of PCV2 ORF2 and 2-50% w / v of inactivated concentrated Mhyo culture as Mhyo per milliliter. Methods for quantifying these immunogens are well known in the art and can also be based on ELISA-based quantification against specific standards. Methods for quantifying these immunogens are well known in the art and can also be based on ELISA-based quantification against specific standards.

[0067] The combination vaccines according to the present invention can advantageously be combined with one or more other antigens or immunogens, which are replicating or non-replicating, whole or disrupted. Thus, in one embodiment, the combination vaccines according to the present invention can contain at least one additional antigen or immunogen. The combination vaccines according to the present invention can contain at least one additional antigen or immunogen. in an amount capable of inducing a protective immune response against those related diseases in animal targets as described above.

[0068] The additional antigen or immunogen is an attenuated form of a microorganism that is pathogenic to pigs. Alternatively, it is a non-replicating antigen or immunogen derived from a microorganism that is pathogenic to pigs. The microorganism can be any virus, bacterium, parasite, fungus, rickettsia, protozoan and / or parasite that is pathogenic to pigs. Examples of such microorganisms that are pathogenic to pigs include pseudorabies virus, porcine parvovirus, porcine cholera virus, porcine influenza virus, foot-and-mouth disease virus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine respiratory coronavirus, vesicular stomatitis virus, Lawsonia intracellularis, Actinobacillus pleuropneumoniae Brachyspira, Escherichia coli (E. coli), Haemophilus Streptococcus, Salmonella, Clostridia, Pasteurella Erysipelothrix, Leptospira, Bordetella, Toxoplasma, Isospora and Trichinella. Preferred additional antigens or immunogens include Lawsonia intracellularis, Actinobacillus pleuropneumoniae, Haemophilus parasuis, Brachyspira hyodysenteriae Examples include. Lawsonia intracellul aris), Actinobacillus pleuropneumoniae), Haemophilus parasuis), Brachyspira one or more from Salmonella choleraesuis, Haemophilus parasuis, Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae (Mhyo), Brachyspira hyodysenteriae, and porcine influenza virus are listed.

[0069] The observed effects of the mixed vaccine according to the present invention are as follows. For Mhyo it is the prevention or reduction of lung lesions caused by Mhyo, such as consolidated pneumonia and chronic respiratory diseases. In the case of Mhyo, the most reliable measure of vaccine efficacy is the reduction in the lung lesion score after Mhyo challenge infection. Such lesions are typically scored during autopsy based on the macroscopic evaluation of lung consolidation according to the Goodwin scale (Goodwin et al., 1969, J. Hyg. Camb. , vol. 67, p. 465-476). This scale ranges from zero to a maximum of 55 points / animal for the completely affected lung.

[0070] For PCV2, it is the prevention or reduction of clinical signs of debilitation or ill thrift, the presence of macroscopic and microscopic lesions characteristic of the disease, and the presence of viral antigen or DNA in microscopic lymphoid lesions. In the case of PCV2, the most reliable measure of vaccine efficacy is the test for the presence of viral nucleic acid by qPCR in serum, fecal swab material, nasal swab material, inguinal lymph nodes, mesenteric lymph nodes, tonsils, and lungs. The induction of antibodies against PCV2 after vaccination is correlated with protection.

[0071] In a preferred embodiment, the mixed vaccine is for pigs. The term "pig" means an animal of the family Suidae, preferably an animal of the genus Sus, also commonly referred to as a pig. Examples of species include wild or domestic pigs, boars, wild boars, babirusas or warthogs. This includes, for example, pigs designated by any name indicating gender or age, such as , sows, queens, wild boars, barrows, hogs, gilts, weaners or piglets. Further, the term pig refers to any type of swine animal, such as a breeding or fattening type, and any parent stock of any of these types.

[0072] The combination vaccine according to the invention can be formulated in various ways as described herein.

[0073] In one embodiment, the combination vaccine according to the invention is provided as a ready-to-use formulation, i.e. a formulation in which all components of the vaccine are pre-mixed so that the combination vaccine can be used directly for vaccination without requiring further mixing or reconstitution steps.

[0074] In another embodiment, the combination vaccine according to the invention can be manufactured from a kit of parts comprising at least two containers, where one container contains all components of the combination vaccine according to the invention except the Myho immunogen and one container contains the Myho immunogen. The PCV or Myho immunogen can be provided, for example, in lyophilized form or as a sterile suspension, such as an aqueous suspension. The lyophilized form can be, for example, a lyophilized cake in a container such as a bottle, but can also be a Riosphere as applied

[0075] Therefore, the combination of the components of the parts kit is one of the embodiments of the combined vaccine according to the present invention. The contents of said at least two containers can be reconstituted in situ immediately before use, i.e., before vaccination. When reconstituting the non-replicating immunogen from Mhyo, a complete combined vaccine according to the present invention is formed. This is also referred to as "on-the-spot" mixing or "field-side" mixing of the vaccine. Therefore, in another aspect, the present invention relates to a parts kit comprising at least two containers, wherein one container contains a non-replicating immunogen from PCV2 in an oil-in-water emulsion comprising squalane, vitamin E-acetate and silica, and one container contains a non-replicating immunogen from Mhyo. In another embodiment, the immunogens of both PCV2 and Mhyo are provided in one container, for example, as an aqueous solution or dispersion, or in lyophilized form, and optionally, it further contains silica and is reconstituted before use with the components of a second container containing (other) adjuvant in the form of an o / w emulsion. The parts kit and its components according to the present invention can include any of the embodiments (preferred or otherwise) described in the present specification with respect to the combined vaccine according to the present invention, or any combination of two or more of those embodiments of the combined vaccine according to the present invention. Therefore, in another aspect, the present invention is

[0076]

[0077]

[0078]

[0079] ·Preparing an aqueous phase containing non-replicating immunogens from PCV2 and Mhyo, and to ·Mixing the aqueous phase with an oil emulsion containing squalane, vitamin E -acetate and silica to form an oil-in-water emulsion relates to a method for producing a combined vaccine according to the present invention, comprising the above.

[0080] In another aspect, the present invention relates to ·Preparing an aqueous phase containing non-replicating immunogens from PCV2, ·Mixing the aqueous phase with an oil emulsion containing squalane, vitamin E -acetate and silica to form an oil-in-water emulsion, and ·Mixing the oil-in-water emulsion with non-replicating immunogens from Mhyo relates to a method for producing a combined vaccine according to the present invention, comprising the above.

[0081] In another aspect, the present invention relates to ·Preparing non-replicating immunogens from Mhyo in a lyophilized form, ·Preparing an aqueous phase containing non-replicating immunogens from PCV2, ·Mixing the aqueous phase with an oil emulsion containing squalane, vitamin E-acetate and silica to form an emulsion, and ·Reconstituting the lyophilized non-replicating immunogens from Mhyo with the mixture of the aqueous phase and the oil emulsion relates to a method for producing a combined vaccine according to the present invention, comprising the above. In yet another aspect, the present invention relates to

[0082] ·Preparing non-replicating immunogens from PCV in a lyophilized form, ·Preparing an aqueous phase containing non-replicating immunogens from Mhyo and silica, ·Mixing the aqueous phase with an oil emulsion containing squalane, vitamin E-acetate and silica · mixing the aqueous phase with an oil emulsion comprising squalane and vitamin E-acetate, and and · reconstituting the lyophilized non-replicating immunogen from PCV with the mixture of the aqueous phase and the oil emulsion relates to a method for producing a mixed vaccine according to the present invention, comprising At various times in these methods, additional steps may be added, for example for additional processing, such as purification or storage. The production method may also include mixing with additional antigens or immunogens,

[0083] or pharmaceutically acceptable excipients, such as stabilizers or preservatives. As described, the mixed vaccine according to the present invention that can be produced by the method according to the present invention is advantageously used for intradermal administration to pigs to protect against infection by PCV2 and Mhyo and / or diseases associated with infection by PCV2 and Mhyo. Therefore, in another aspect, the present invention relates to an oil-in-water emulsion comprising squalane, vitamin E-acetate, silica, and a non-replicating immunogen from PCV2 and Mhyo for use in intradermal vaccination of animals such as pigs against PCV2 and Mhyo. In another aspect, the present invention relates to the use of a non-replicating immunogen from PCV2 and Mhyo for the production of a mixed vaccine for animals such as pigs, characterized in that the vaccine is an oil-in-water emulsion comprising squalane, vitamin E-acetate and silica.

[0084]

[0085]

[0086] ​​​​​​​​​​​​

[0087] The mixed vaccine according to the present invention can be applied to the vaccination of pigs against PCV2 and Mhyo. It can be applied.

[0088] Thus, in another aspect, the present invention provides a water-in-oil emulsion comprising squalane, vitamin E-acetate, silica and a non-replicating immunogen from PCV2 and Mhyo, which is administered intradermally to an animal. It relates to a method for vaccinating animals such as pigs against PCV2 and Mhyo. Alternatively, in a similar embodiment, the present invention relates to a method for vaccinating animals such as pigs against PCV2 and Mhyo by intradermally administering the mixed vaccine according to the present invention to the animal. It relates to a method for vaccination.

[0089] Therefore, the mixed vaccine according to the present invention is typically administered intradermally into the skin of an animal, i.e., by intradermal administration. This can be achieved by various methods, for example, by using a conventional syringe and hypodermic needle. Alternatively, parenteral administration can be performed by any method of needle-free injection that delivers the vaccine by means of an intradermal applicator such as the IDAL (registered trademark) applicator of MSD Animal Health. It can be achieved by using a conventional syringe and hypodermic needle. It relates to a method for vaccination.

[0090] Thus, the mixed vaccine according to the present invention is typically administered intradermally into the skin of an animal, i.e., by intradermal administration. This can be achieved by various methods, for example, by using a conventional syringe and hypodermic needle. Alternatively, parenteral administration can be performed by any method of needle-free injection that delivers the vaccine by means of an intradermal applicator such as the IDAL (registered trademark) applicator of MSD Animal Health. This can be achieved by various methods, for example, by using a conventional syringe and hypodermic needle. Alternatively, parenteral administration can be performed by any method of needle-free injection that delivers the vaccine by means of an intradermal applicator such as the IDAL (registered trademark) applicator of MSD Animal Health. It can be achieved by any method of needle-free injection that delivers the vaccine by means of an intradermal applicator such as the IDAL (registered trademark) applicator of MSD Animal Health. It can be achieved by any method of needle-free injection that delivers the vaccine by means of an intradermal applicator such as the IDAL (registered trademark) applicator of MSD Animal Health.

[0091] For suitability for intradermal administration, the volume of the animal dose of the mixed vaccine according to the present invention is typically from 0.05 to 1.0 ml per animal, preferably from 0.1 to 0.5 ml, more preferably about 0.2, 0.3 or 0.4 ml, and most preferably about 0.2 ml per animal dose (the latter being more preferred). It is typically from 0.05 to 1.0 ml per animal, preferably from 0.1 to 0.5 ml, more preferably about 0.2, 0.3 or 0.4 ml, and most preferably about 0.2 ml per animal dose (the latter being more preferred). It is more preferably about 0.2, 0.3 or 0.4 ml, and most preferably about 0.2 ml per animal dose (the latter being more preferred). It is most preferably about 0.2 ml per animal dose (the latter being more preferred).

[0092] The dosing regimen for the method of vaccinating a target pig according to the present invention is a single dose or in multiple doses, or is by a method adapted to the actual practice of pig farming can be.

[0093] Optionally, for the animal target, at a later time, a second or further administration, a so-called booster vaccination, of the combined vaccine according to the present invention can be carried out. However, the combined vaccine according to the present invention is optimized such that a single vaccination dose is generally sufficient to obtain immune protection during an important period of the animal's life, for example, during the fattening stage of pigs up to 6 months of age period. Therefore, in a preferred embodiment, the combined vaccine according to the present invention is administered only once per animal target i.e., it is a single-dose (single administration) vaccine.

[0094] Preferably, in order to further reduce stress on the animal and reduce labor costs, the regimen of the vaccination method is incorporated into the existing vaccination schedule of other vaccines that the target pig may require. These other vaccines can be administered in a manner compatible with their registered uses, simultaneously, in combination, or in a sequential manner. Therefore, in one embodiment of the method of vaccinating pigs according to the present invention, the combined vaccine according to the present invention is administered in combination with another porcine vaccine

[0095] When the target animal is a pig, the target pigs for vaccination according to the present invention can be of any age that is susceptible to the vaccine species, and / or of any age that is susceptible to the diseases or infections that the vaccine protects against. Therefore, in one embodiment of the method of vaccinating pigs according to the present invention, the combined vaccine according to the present invention is administered to young pigs ​ The vaccine is administered to piglets, i.e., pigs at about 2 months of age. Alternatively, the combination vaccine according to the present invention is administered to adult pigs, i.e., pigs at about 6 months of age.

[0096] Since the prevalence of Mhyo and PCV2 is high and vaccines against one or more of these pathogens are widely used, a large number of female pigs will be seropositive for antibodies against one or more of Mhyo and PCV2. As a result, piglets inoculated with colostrum from such female pigs may become MDA+ (positive for maternally-derived antibodies). This does not impair the effectiveness of the combination vaccine according to the present invention at all, because it is effective even in MDA+ pigs. Thus, in one embodiment of the method of vaccination according to the present invention, the combination vaccine according to the present invention is administered to MDA+ pigs.

[0097] Administration of the combination vaccine according to the present invention can be applied as either a prophylactic treatment or a therapeutic treatment, or both. This is because it prevents both the establishment and progression of infections caused by Mhyo and PCV2. The use of the combination vaccine according to the present invention will help reduce infections by one or both of Mhyo and PCV2 in pigs in a pig population, farm or a geographical area. Thus, in another aspect the present invention relates to a method for reducing infections by Mhyo and PCV2 or related disease symptoms in pigs, characterized by intradermally administering the combination vaccine according to the present invention to pigs.

[0098]

[0099] Next, the present invention will be described in more detail by the following non-limiting examples.

[0099] Example Example 1: Production of the combined vaccine The combined vaccine according to the present invention was produced as follows.

[0100] This oil-in-water emulsion at double concentration was produced according to the subsequent process steps below.

[0101] · Weighed out the required amounts of vitamin E acetate and squalane and combined them in a beaker. did.

[0102] · The vitamin E acetate / squalane mixture was homogenized at room temperature by low-energy mixing (magnetic stirrer). did.

[0103] · Weighed out the required amount of polysorbate 80 and added it to the homogenized vitamin E acetate / squalane mixture. did.

[0104] · The combined mixture was homogenized again at room temperature by low-energy mixing.

[0105] · The homogenized mixture was filter-sterilized through a 0.2 micrometer filter (Pall, Ultipor (trademark) N66).

[0106] · Weighed out the required amount of (heat-sterilized) silica and added it to the homogenized mixture, and then the combined mixture was homogenized again at room temperature by low-energy mixing.

[0107] · The mixture was heated to 65 - 75 °C.

[0108] · Sterile water for injection was heated to 65 - 75 °C.

[0109] · The heated oil phase and water were mixed at high energy using an N18 rod and an Ultra Turrax Pre-mixing was carried out for 5 to 15 minutes using a Nelgy mixture. The temperature decreased from 65 °C to 55 °C .

[0110] · The pre-mixture was passed through a Microfluidizer (trademark) three times at 800 bar . The temperature was maintained below 50 °C with a cooling spiral

[0111] The integrity and homogenization level of the final oil-in-water emulsion were examined by optical microscopy . Furthermore, the pH (7.34) and osmotic pressure (221 mOsm / kg) were also examined. The particle size measurements are shown below: D100 = 300 nm; D99 = 250 nm; D90 = 200 nm; and D50 = 130 nm

[0112] The aqueous phase (2-fold concentration) was prepared by taking the respective required amounts of each of the following non-replicating immunogens : Mhyo: 6% v / v of 10-fold concentrated inactivated culture and PCV: 50 μg ORF 2

[0113] Next, both concentrated compositions (oil-in-water emulsion containing an adjuvant and aqueous phase containing an immunogen ) were mixed at room temperature by low-energy mixing at a volume ratio of approximately 50:50

[0114] Using the method described above, the following vaccine adjuvant compositions were prepared as oil-in-water (o / w) emulsions (all percentages are % w / v). When preparing the oil-in-water emulsion, in some compositions, aluminum hydroxide in the indicated amount (as a 2-fold concentrate) was added together with polysorbate 80 and squalane .

Table 1

[0115] ​​Example 2: Efficacy of a PCV2 / Mhyo ID formulation in pigs against Mhyo challenge Efficacy of the agent The efficacy of vaccination with an Mhyo intradermal (ID) formulation (0.2 ml, inoculated with I DAL (registered trademark) vaccine on the right side of the neck) was tested in specific pathogen-free (S PF) piglets (ToJaPigs). The formulation was produced by various treatments of Mhyo immunogen and formulated at 5 PCVU / ml (about 25% w / v) of inactivated Mhyo culture. Animals at 3 weeks of age were vaccinated according to the following scheme (Table 2). Four weeks after vaccination, all animals were infected with Mhyo. All animals were challenged with 10 and 10 9 and 10 9 CCU / ml of 10 ml of the tracheal Mhyo strain 98 at 7 weeks of age (i.e., 4 weeks after vaccination) for 2 consecutive days. Three weeks after challenge, the animals were sacrificed and the degree of Mhyo-induced sclerosing pneumonia was scored according to Goodw in (maximum score: 55). [Table 2]

[0116] Results: The efficacy of vaccination was determined by the lung lesion score (LLS, mean). This was recorded for each pig and compared with the unvaccinated control group. [Table 3]

[0117] In neither of the vaccines tested in groups 1 and 2 and compared with the control group was a significant decrease in the lung lesion score obtained. On the contrary, the lung lesion core was unvaccinated It was similar to the inoculation control group.

[0118] Therefore, the combination of adjuvant aluminum hydroxide, squalane, vitamin E acetate and silica combined with the immunogens of PCV2 and Mhyo was also not suitable for safe and effective intradermal administration, as was the combination of the commercially available PC V vaccine Porcilis® PCV ID and silica as an adjuvant.

[0119] Example 3: Efficacy of a PRRS vaccine reconstituted in a PCV2 / Mhyo ID formulation in pigs The purpose of this study was to compare the safety and serological efficacy of various PCV2 and / or Mhyo vaccines containing Aerosil 200 or Aerosil 380 reconstituted with Porcilis® PRRS when administered intradermally (ID) to the necks of 5-week-old piglets. The piglets were assigned to the treatment groups shown below ( Table 4). The piglets were intradermally vaccinated at approximately 5 weeks of age. As described below, 10 TCID 50 50 4.5 50 50 virus contained in Porcillis PRRS was used to vaccinate the piglets in groups 4 and 5.

Table 4

[0120] · A single dose ( 0.2 ml) of a vaccine formulated with PCV2 (10,000 AU / ml; approximately 80 μg / ml) + M. Hyo (10 PCV U / ml) and adjuvant Aerosil 200 was used to intradermally vaccinate the piglets in group 4.

[0121] · Piglets in Group 5 were inoculated intradermally with a single dose (0.2 ml) of the vaccine formulated with PCV2 (10,000 AU / ml) + M. Hyo (10 PCVU / ml) and adjuvant Aerosperse 380.

[0122] · Piglets in Group 6 were inoculated intradermally with a single dose (0.2 ml each) of the vaccines Porcilis® PCV ID + Porcilis® ) PRRS (unmixed).

[0123] · Piglets in Group 7 were not vaccinated (negative control group). All animals were examined for injection site reactions.

[0124] All piglets were observed daily for clinical signs after vaccination. Body temperature was measured and serum samples were collected from all animals. Samples were tested for antibodies against PCV2, Mycoplasma hyopneumoniae ( M. Hyopneumoniae) and PRRSV.

[0125] Results: After vaccination, the body temperatures of all groups were equivalent. On the day of vaccination, all animals had equivalent PCV2 antibody titers. The PCV2 antibody titers remained at a constant level in all groups until the end of the study. The antibody titers in control group 7 decreased over time. On the day of vaccination, all animals were negative for PRRS antibodies. All groups showed 0 - 20% PRRS responders. Control group 7 remained negative. At the start of the study, all animals were serologically negative for Mycoplasma hyopneumoniae. All groups showed 0 - 20 % responders at SD21. At SD28, most groups showed positive animals. Control group 7 remained negative. ​ did not show a mycoplasma hyopneumoniae positive response. IgM antibody responses (predicting vaccination) against PCV2 and Mhyo are shown in Tables 5 and 6.

Table 5

Table 6

[0126] From this study, it can be concluded that none of the vaccinated groups showed an acceptable antibody response against PCV2 or Mhyo. Therefore, adjuvant E -rosil A380 or Aerosil A200, when reconstituted with PRRS and PCV2 as well as Mhyo immunogens, was not suitable for safe and effective intradermal administration in pigs.

[0127] Example 4: Efficacy of a PRRS vaccine reconstituted in an Mhyo ID formulation against Mhyo challenge infection in pigs The efficacy of vaccination with an Mhyo intradermal (ID) formulation mixed with PRRS (A212D, 10 5.1 TCID50 / dose) against Mhyo challenge infection was tested in SPF piglets. The formulation was manufactured using various adjuvants. According to the following scheme (Table 7), the animals were ID vaccinated (0.2 ml) on the right side of the neck at 3 weeks of age. Three weeks after vaccination, all animals were infected with Mhyo.

Table 7

[0128] ​​​​​All animals were infected with 10 ml of intratracheal Mhyo strain 98 at 6 weeks of age for 2 consecutive days with 9 and 8 CCU respectively.

[0129] Results: The efficacy of vaccination was determined by the lung lesion score (LLS, mean). This was recorded for each pig and compared to the unvaccinated control group.

Table 8

[0130] Intradermal administration of an adjuvant composition of aluminum hydrogel, squalane, and vitamin E-acetate in combination with Mhyo and PRRS immunogens, as in reference composition 3, resulted in a very low, and thus unacceptable, reduction rate of lung lesion score of 22%, compared to 74% in the positive control group using a commercial Mhyo vaccine. Therefore, the combination of the adjuvants aluminum hydrogel, squalane, and vitamin E-acetate with the immunogens was not suitable for safe and effective intradermal administration.

[0131] Example 5: Efficacy of PCV2-Mhyo ID formulation in SPF pigs against Mhyo challenge infection According to the following scheme (Table 9), a group of 12 pigs was vaccinated intradermally at 3 weeks of age (+ / - 3 days). Group 17 was not vaccinated and was used as a Mhyo challenge control. Four weeks after vaccination, all animals were infected with a virulent Mhyo strain. Three weeks after challenge, all animals were necropsied for lung lesions. Blood samples were taken before vaccination, before challenge, and postmortem.

Table 9

[0132] Experimental procedure Serology Immediately before vaccination (T = 0, 3 weeks of age), immediately before challenge (T = 4, 7 weeks of age) and at necropsy (T = 7, 10 weeks of age), blood samples (jugular vein) were collected. Samples were transported at ambient temperature. Serum was obtained in duplicate from the blood samples. The presence of relevant antibodies in all serum samples was determined by standard methods in ELISA tests for M. hyo and PCV.

[0133] Clinical observation and rectal temperature Clinical observations were made and rectal temperatures were measured immediately before vaccination, 4 hours after vaccination and 1 day and 2 days after vaccination. Observations and temperatures were recorded.

[0134] Postmortem examination At the end of this experiment, 4 weeks after challenge, the pigs were sedated by electric shock and then euthanized by exsanguination. Lung lesions were scored according to Goodwin.

[0135] Results: No unacceptable injection site reactions were observed. No unacceptable temperatures were observed.

[0136] The efficacy of vaccination was determined by the lung lesion score (LLS, mean). This was recorded for each pig and compared with the unvaccinated control group. The results are shown in Table 10. [Table 10]

[0137] ​​​​The data of the lung lesion score showed that groups 11, 12 (the composition of the present invention) and 13 (reference composition) 2) showed acceptable scores. This is because the reduction rate compared to the unvaccinated control group (group 17) exceeded 45% (which was used as the cut-off value for substantial efficacy). On the other hand, groups 14 and 15 showed unacceptable scores . This is because the LLS in group 15 was much higher than that of the unvaccinated control group, and the LLS in group 14 only decreased by 20% compared to the negative control group . Therefore, the vaccine composition of the present invention containing a combination of squalane, vitamin E-acetate and silica adjuvant

[0138] was able to give an acceptable lung lesion score indicating effective vaccination.

[0139] Example 6: Efficacy of D formulation of PCV2-Mhyo I in SPF pigs against PCV challenge infection Ten piglets were assigned to each of five treatment groups of ten piglets, and when they reached about 3 weeks of age they were vaccinated intradermally. Piglets in groups 1-3 were vaccinated with the vaccines described below . Group 4 was vaccinated with Porcilis® PCV ID and Porcilis® M Hyo ID ONCE as a positive control group . Group 5 did not receive vaccination (negative control). Three weeks after vaccination (6 weeks of age), all animals were challenged using 5.0 log10 TCID5 0 / mL of the wild-type PCV2b challenge virus strain I12 / 11 by intranasal application (3 ml per nostril). The treatment plan is shown in Table 11 below. Three weeks after the challenge, all animals were necropsied and PCV2​​​ The inguinal lymph nodes, mesenteric lymph nodes, tonsils and lungs were sampled for the detection of .

[0140] All piglets were observed daily after vaccination for clinical signs. Serum samples were collected on the day of vaccination as well as at SD14, SD20, SD35 and SD42 (during necropsy). Samples were tested by qPCR for antibodies against PCV2 virus nucleic acid. Fecal swabs were collected from all animals at SD20, 35 and SD41 and tested by qPCR for PCV2 virus nucleic acid.

Table 11

[0141] Treatment Dosage and administration Vaccination was performed by the intradermal route (0.2 ml) on the right side of the neck. Group 21 was vaccinated twice on the right side. The challenge was performed by the intranasal route using a MAD applicator at 6 ml and 3 ml per nostril.

[0142] Test system Only healthy animals were used. They were examined before vaccination (absence of general appearance and clinical abnormalities or diseases) to exclude non - healthy animals. Immediately before vaccination, all animals were individually tagged with ear tags. All pigs were observed daily for clinical signs of disease. The observations consisted of systemic reactions such as anorexia, exercise aversion, tendency to lie down, lethargy or drowsiness, shivering, bristling, edema (especially around the eyes), vomiting and diarrhea and dyspnea.

[0143] Experimental method Blood sample collection ​​​​​​​​On the vaccination date, 1 day before challenge, 2 weeks after challenge, and necropsy date, blood samples were collected from all animals. A minimum of 4 ml and a maximum of 8 ml of blood were collected per animal. This was performed individually from all pigs according to standard procedures. Blood samples were collected without adding anticoagulants.

[0144] Fecal swab Fecal swabs were taken 1 day before challenge infection, 2 wpc, and 1 day before necropsy. The swab sticks were placed in a medium containing antibiotics.

[0145] Postmortem examination Three weeks after challenge, the animals were transferred to the necropsy room. They were anesthetized using an electrocution device and exsanguinated according to standard procedures. The carcasses of the animals were disposed of according to standard procedures. During necropsy, the animal's body was opened and the internal organs were examined in situ with particular attention paid to the following organs: lungs, inguinal lymph nodes and mesenteric lymph nodes, tonsils, thymus, spleen, liver, and kidneys . Samples were then taken from the tonsils, lungs, mesenteric lymph nodes, and inguinal lymph nodes and divided into two parts. One was for analysis by freezing and PCV2 qPCR, and the other was for fixation and subsequent (immuno)histochemical analysis. Serum was prepared from the clotted blood samples and aliquots (e.g., 2 × 0.8 ml) were filled

[0146] Sample processing with. The samples were not heat inactivated. Fecal samples were prepared from the swabs and aliquots (e.g ., 2 × 0.8 ml) were filled. The samples were stored at -15°C or below until use . The time from sample collection to storage did not exceed 36 hours (serum) or 48 hours (swabs) . None of the samples exceeded this time limit.

[0147] PCV2 antibody ELISA Serum was tested for antibodies against PCV2 according to standard procedures. Briefly, serial diluted serum samples were incubated on microtiter plates coated with baculovirus-expressed PCV2 ORF2 antigen. After removing the serum, all wells were incubated with a fixed amount of biotin-labeled PCV2-specific monoclonal antibody (MoAb). Then the bound MoAb was incubated with peroxidase-conjugated streptavidin, and then chromogenic detection was performed. The titer was defined as the reciprocal of the serum dilution at which the absorbance value was equal to the absorbance value at 50% of the maximum absorbance of the test. The results were expressed as log2 titers. Titers less than 2.0 log2 were considered negative. For negative samples, a value of 1.0 log2 was used for calculation purposes.

[0148] Quantitative PCR Quantitative PCR (qPCR) for PCV2 nucleic acid was performed on all sera, fecal swabs, and 10% tissue homogenates of tonsils, lungs, mesenteric lymph nodes, and inguinal lymph nodes according to standard procedures. Briefly, DNA was extracted from the samples using a commercially available kit. The PCV2 genomic DNA in each sample was quantified by polymerase chain reaction (PCR) using a probe and primers specific for PCV2-ORF2. The cycle number at which specific fluorescence exceeded the threshold correlated with the cycle number for a set of samples containing known amounts of PCV2-ORF2-containing plasmid. The results were expressed as log copies / μl (log c / μl) of the extracted DNA. 1.00 log 10 10 c / μl)​​​​​​​​10 c / Values less than μl were considered negative and 0.00 log was used for calculation purposes 10 as c / μl.

[0149] Immunohistology (IHC) Samples of tonsils and lymph nodes were prepared for histological examination. The samples were fixed in 10% formalin, paraffin-embedded, and immunohistochemical examination was performed for the detection of PCV2 antigen on slides using anti-PCV2 rabbit serum as the primary antibody and Envis ion+ (DAKO, Denmark) as the detection system according to the manufacturer's instructions used. The slides were counterstained with hematoxylin. Microscopic examination was performed. For tonsils and lymph nodes, characteristic brown staining was scored on the following ordinal scale.

[0150] 0: No specific positive staining cells were observed.

[0151] 1: Scattered (single) positive staining cells are present in less than 10% of the follicles.

[0152] 2: Positive staining (single) cells are observed in 10 - 50% of the follicles, or a localized aggregation of positive staining cells exceeding 15 is observed in less than 10% of the follicles.

[0153] 3: Specific staining in more than 50% of the follicles.

[0154] The results were recorded as the total score, which is the sum of the scores of individual tissues.

[0155] Results: PCV2 serology: At the start of measurement in SD1, the log2 titers of all groups were 4 - 5. The positive control ( Group 21) showed a log2 titer of 10 between SD34 and SD41. In the same week, Group 18 ​, the log2 titers at 19 and 20 were 8 to 10. The unvaccinated negative control group 22 showed a log2 titer of less than 4.

[0156] Amount of PCV2 virus in serum (qPCR serum) Between SD0 (start of measurement) and SD19, in all groups, the virus amount could not be detected (log10 c / μl = 0).

[0157] At SD34, the virus amount in the positive control (group 21) increased to about 1.0 log10 c / μl and further increased to about 1.6 at SD41.

[0158] At SD34, the virus amount in the unvaccinated negative control (group 22) increased to about 4.1 log10 c / μl and slightly decreased to about 3.6 at SD41.

[0159] The virus amounts in groups 18, 19 and 20 were 1.3 - 1.8 log10 c / μl at SD34 and 1.6 - 2.1 at SD41.

[0160] Amount of PCV2 virus in fecal swab (qPCR fecal swab) At SD19 (start of measurement), the virus amount could not be detected in all groups (log 10 c / μl = 0). At SD34, the virus amount in the positive control (group 21) increased to about 1.5 log10 c / μl and further increased to about 2.2 at SD41.

[0161] The virus amount in the unvaccinated negative control (group 22) increased to about 3.6 log 10 c / μl at SD34 and remained basically constant until SD41.

[0162] The virus amounts in groups 18, 19 and 20 were within the range of the positive control group (at SD34 It was about 1.5 to 1.8, and about 1.8 to 2.2 in SD41.

[0163] Safety No unacceptable injection site reactions were observed. The maximum average temperature rise was 0.4 °C at T = 0 + 4 hours Thus, the vaccine composition of the present invention containing a combination of squalane, vitamin E-acetate and silica was able to show that it produced acceptable results with respect to vaccine efficacy without causing unacceptable site reactions .

[0164] Example 7: Use of different pharmaceutical grade silicas constituting a mixed vaccine In this example, various different types of silica [all pharmaceutical grade (colloidal) amorphous silica] have been shown to be useful for producing the mixed vaccine according to the present invention . The method for producing the vaccine was almost identical to that of Example 1. However, in some vaccines silica was added only after passing through a Microfluidizer (trademark). This had no substantial effect on the final composition. All silica was generally distributed in the final composition having an average particle size of about 120 nm. However, when silica was added after passing through a Microfluidize r (trademark), a very small proportion of the total amount of silica was present as large aggregates (solid particles) exceeding 10 μm (maximum 200 μm). However, this did not adversely affect the properties of the vaccine. Data on various compositions are shown in Table 12 .

Table 12

[0165] ​Other silicas suitable for use in the present invention include Aerosil (registered trademark) 90, Aerosil (registered trademark) 130, Aerosil (registered trademark) 150, A erosil (registered trademark) 200F, Aerosil (registered trademark) 255, Aerosi l (registered trademark) OX 50, Aerosil (registered trademark) TT600 and Aerope rl (registered trademark) 300 / 30.

[0166] Conclusion Examples 1 to 7 show that among the tested compositions, only the novel adjuvant combination of squalane, vitamin E-acetate and silica results in acceptable results regarding vaccine efficacy against PCV2 and Mhyo infections without causing unacceptable local reactions. Therefore, a mixed vaccine against PCV2 and Mhyo using a non-replicating immunogen of porcine circovirus type 2 and Mycoplasma hyopneumoniae for safe and effective intradermal administration can be provided using this adjuvant composition. ​​​​​​

Claims

1. A combined vaccine comprising a non-replicating immunogen of porcine circovirus type 2 and a non-replicating immunogen of Mycoplasma hyopneumoniae, which is an oil-in-water emulsion containing squalane, vitamin E-acetate and silica. The combined vaccine is characterized by being an oil-in-water emulsion containing squalane, vitamin E-acetate and silica.

2. The combined vaccine according to Claim 1, containing squalane in an amount of 1 to 15% w / v.

3. The combined vaccine according to Claim 1 or 2, containing vitamin E-acetate in an amount of 2 to 20% w / v.

4. The combined vaccine according to any one of Claims 1 to 3, containing an emulsifier having an HLB value of 8 to 20.

5. The combined vaccine according to Claim 4, wherein the emulsifier is polysorbate 80.

6. The combined vaccine according to Claim 4 or 5, wherein the emulsifier is present in an amount of 0.5 to 10% w / v.

7. The combined vaccine according to any one of Claims 1 to 6, containing silica in an amount of 0.02 to 2% w / v.

8. The combined vaccine according to any one of Claims 1 to 7, wherein the non-replicating immunogen of porcine circovirus type 2 is a recombinant expressed protein encoded by the ORF2 gene of porcine circovirus type 2.

9. The combined vaccine according to any one of Claims 1 to 8, wherein the non-replicating immunogen of Mycoplasma hyopneumoniae contains killed whole Mycoplasma hyopneumoniae.

10. The combined vaccine according to any one of Claims 1 to 9, for use in the prophylactic treatment of animals against infection by porcine circovirus type 2 and infection by Mycoplasma hyopneumoniae, characterized in that the combined vaccine is administered intradermally to the animal.

11. The combined vaccine for use according to Claim 10, wherein the volume of the combined vaccine administered intradermally to the animal is 0.1 to 0.5 ml.

12. For the production of the combined vaccine according to any one of Claims 1 to 9 for prophylactically treating animals against infection by porcine circovirus type 2 and infection by Mycoplasma hyopneumoniae, a non-replicating immunogen of porcine circovirus type 2 and a non-replicating immunogen of Mycoplasma hyopneumoniae ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ The use, characterized in that, in the original use, the combined vaccine is administered intradermally to an animal. Use. **Claim 13** A method for prophylactic treatment of an animal against infection by porcine circovirus type 2 and infection by Mycoplasma hyopneumoniae, by administering the combined vaccine according to any one of claims 1 to 9 intradermally to the animal. **Claim 14** An adjuvant composition for formulating a non-live vaccine, characterized in that it is an oil-in-water emulsion containing squalane, vitamin E-acetate and silica. ​

Images